Sequence Listing
The Sequence Listing submitted via EFS-Web as ASCII compliant text file format (.txt) filed on Jan. 25, 2017, named “SequenceListing_ST25”, (created on Jan. 22, 2016, 24 KB), is incorporated herein by reference. This Sequence Listing serves as paper copy of the Sequence Listing required by 37 C.F.R. § 1.821(c) and the Sequence Listing in computer-readable form (CRF) required by 37 C.F.R. § 1.821(e). A statement under 37 C.F.R. § 1.821(f) is not necessary.
Field of the Invention
This invention relates to double stranded RNA and compositions containing double stranded RNA (dsRNA) to reduce the fitness and/or increase mortality of Diaphorina citri. (Asian citrus psyllid, Hemiptera: Liviidae) thereby reducing infestation of D. citri on citrus plants, and thereby reducing transmission of plant pathogens for which D. citri is a vector. One example of such microorganisms are Candidatus Liberibacter species. The invention also relates to methods of reducing the fitness and/or increasing mortality of D. citri by applying dsRNA as topical sprays, soil treatment, or other delivery methods to plants on which D. citri feeds. The invention also relates to genetically altered plants that express the dsRNA described herein; and to genetically altered microorganisms that express the dsRNA described herein.
Description of Related Art
Huanglongbing (HLB) (also called “citrus greening disease”) is the most serious disease that threatens citrus crops worldwide. It is especially devastating to the U.S. and Florida citrus industries. The causative agents of HLB are Candidatus Liberibacter species of bacteria. This includes Candidatus Liberibacter africanus (CLaf), Candidatus Liberibacter asiaticus (CLas), and Candidatus Liberibacter americanus (CLam), however, until 2015 only CLas was found in U.S. The CLas and CLam bacteria are transmitted from plant to plant via Asian citrus psyllid (ACP; D. citri). The CLaf bacteria is transmitted from plant to plant via the psyllid Trioza enytreae. This disease is devastating the citrus industry in Florida because no effective treatments currently exists. Furthermore, both the causative pathogen and the insect vector have spread to other parts of the U.S. (California, Texas, Arizona) as well as to other citrus-producing countries/regions (Brazil, Asia, Middle East, China). Further, Murraya paniculata and other plants also host CLam and/or CLas. D. citri feed on these infected plants and carry the bacteria to non-infected plants. A list of these host plants could be found in freshfromflorida.com/content/download/24041/486974/hostlist.pdf. As such, the need exists for a composition and methods to stop the transmission of the disease. One approach to preventing disease transmission is to increase the mortality of ACP, the vector for the bacteria. Another approach is to reduce the fitness of ACP. Reducing ACP populations, by increasing mortality or reducing fitness, is the goal, thereby reducing transmission of the bacteria and the disease. One approach for reducing ACP populations, while not reducing beneficial insects, is to use RNAi technology.
Fire, et al. (U.S. Pat. No. 6,506,559) disclose a process of introducing RNA into a living cell to inhibit gene expression of a target gene in that cell. This cellular mechanism was named RNA interference, or RNAi. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical. Specifically, Fire, et al. (U.S. Pat. No. 6,506,559) disclose a method to inhibit expression of a target gene in a cell, the method involves introducing a double-stranded ribonucleic acid, dsRNA, into the cell in an amount sufficient to trigger the RNAi process which leads to inhibition of specific protein translation of the target gene's messenger RNA (mRNA). One strand of the dsRNA trigger has a sequence which corresponds to the nucleotide sequence of the target mRNA. The dsRNA triggers the cell's natural defense mechanism, defined as RNA interference (RNAi), which uses the dsRNA trigger (designed dsRNA herein), to produce the guide strand small interfering RNA (siRNA) which is incorporated into the RNA-induced silencing complex (RISC) which is a multiprotein complex, specifically a ribonucleoprotein, which incorporates one strand of a double-stranded RNA (dsRNA) fragment. The RISC siRNA complex causes degradation of the targeted mRNA, thereby preventing translation into a protein. As used herein, a “trigger” is any dsRNA molecule that causes RNAi activity against a specific gene.
One mechanism of action involves a long trigger (i.e., a long dsRNA) being cleaved by an enzyme called “dicer” to produce several siRNA that are shorter in length than the dsRNA. The size of these smaller siRNA is believed to range from about 19 base pairs to about 25 base pairs, but the most common classes of siRNA contain 21 base pairs or 24 base pairs (Hamilton, et al., 2002 EMBO J., 21:4671-4679). However, others have determined that the siRNA can be shorter than 19 base pairs. See, e.g., Guleda, et al., Geno. Proteomic Bioinfo., 184(6)183-199 (2011). The siRNA molecules are each then incorporated into RISC. The duplex RNA is unwound leaving the anti-sense strand to guide RISC to complementary mRNA for subsequent endonucleolytic cleavage. This results in the reduction of the corresponding protein that would have been made from the targeted and degraded mRNA. Thus, this is commonly referred to as gene-silencing or downregulation.
A few RNAi sequences have been identified and are currently being evaluated for (i) reducing ACP vector capacity to host CLas, and (ii) nymph and/or adult survival. See, e.g., El-Shesheny, et al. (2013) PLoS ONE 8(5): e65392 (doi.org/10.1.3711journal.pone.0065392); and Killiny, et al. (2014) PLoS ONE 9(10): e110536 (doi.org/10.1371/journal.pone.0110536). Li, et al., Insect Sci., 20:31-39 (2013) reviews attempts to use RNAi to control Hemiptera and the overall lack of success of killing Hemiptera via this technique.
One goal of using RNAi is that if one selects a RNA sequence that is relatively unique to the target pest (in this case, D. citri) and which is effective in reducing expression of the target gene such that increased mortality of the target pest occurs, then the treatments can avoid harming beneficial insects, such as pollinators, predators, and parasitoid species. This invention successfully meets these goals in that the dsRNA sequence for ACP trehalase mRNA target (described herein) appears to be unique to ACP and does not harm bees or other pollinators. Trehalase hydrolyzes the disaccharide α,α-trehalose to two molecules of D-glucose. α,α-trehalose is the main sugar found in insect hemolymph (insect blood), and it has a critical role in energy production and biosynthesis of macromolecules including chitin. Trehalase has been purified and characterized from several different insects where it is found in soluble (Tre-1) and membrane-bound (Tre-2) forms encoded by distinct genes. The soluble form is found in hemolymph and the bound form is found in many different insect tissues See, Lee, et al., (2007) Biosci Biotechnol Biochem 71(9);2256-65.
The coding sequence of ACP trehalase (hereinafter “trehalase”) was obtained by automated computational analysis from a D. citri genomic sequence (NW_007377674.1), annotated using the gene prediction method Gnomon (NCBI, Rockville, Md.). See also, Reese, et al. (2013) J. Genomics 2:54-58; doi: 10,7150/jgen.7692. Supporting evidence includes similarity to 16 proteins, and 100% coverage of the annotated genomic feature by RNAseq alignments, including 5 samples with support for all annotated introns. Comparative analysis of the coding sequence of trehalase with other insects genomic sequences indicate that it corresponds to Tre-1 (soluble form of trehalase), as it shares higher homology to other known insects soluble trehalase homologs (Aphis glycines, GenBank: mRNA: JQ246351.1/protein: AFJ00065.1, Nilaparvata lugens, mRNA: H790319.1/protein: ACN85420.1; Locusta migratoria, mRNA: H795020.1/protein: ACP28173,1). See, Reese, et al. (2013) J. Genomics 2:54-58 (doi: 10.715/gen.7692); Hunter, et al. (2009) Open Entomol. 3:18-29; Hunter, et al. (2008) Florida Entomology Society meeting, abstracts, p.11 (flaentsoc.org/2008annmeetabstracts.pdf); and Hunter, et al. (2014) J. Citrus Pathology 1:4.7 (Proc. 3rd Int'l Res. Conf, Huanglongbing, Orlando, Fla.). The cDNA sequence of trehalase is in SEQ ID NO: 26.
A need exists for reducing transmission of CLas and/or CLam to non-infected plants, and in particular, citrus plants. A method of reducing fitness and survival of D. citri (and thereby reducing D. citri infestation) reduces the number of infected D. citri that can transmit HLB pathogens to uninfected plants, thereby slowing down or stopping pathogen transmission and disease spread. Furthermore, any solution to this problem which does not reduce beneficial insects, such as predators and parasitoids of psyllids, as well as beneficial pollinators, will have the added benefits of biological control pressures that will help suppress psyllid populations.